Naphthenic acids, zinc salts

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 November 2017 - 01 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test"

Version / remarks:

Official Journal of the European Union No. L142, 31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Naphthenic acids, zinc salts

EC Number:

234-409-2

EC Name:

Naphthenic acids, zinc salts

Cas Number:

12001-85-3

Molecular formula:

C20H34O4Zn

IUPAC Name:

Naphthenic acids, zinc salts

Test material form:

solid: bulk

Details on test material:

- Description: Zinc salts of naphthenic acids, consisting of an acidic (naphthenic) fraction (70-95%) composed mainly of C8-C20 and 0-3 rings and a non-acidic (petroleum) fraction (5-30%), composed mainly of C12-C22, <10% aromatics, with a boiling range of approximately 160-350°C.
- Starting materials: Naphthenic acids (EC 215-662-8; CAS 1338-24-5); Zinc oxide (EC 215-222-5; CAS 1314-13-2, 7440-66-6)
- Manufacturing method: Zinc oxide and the naphthenic acids, with or without water, are reacted together in a solvent or vehicle, such as base oil to form zinc naphthenate. Sufficient amount of zinc oxide is added to the naphthenic acids to completely neutralise all the naphthenic acids, without significant excess of either starting material. The mixture is heated to complete the reaction then cooled. The amount of zinc oxide is calculated depending on the Total Acid Number of the naphthenic acid starting material. This calculation ensures that no significant excess unreacted zinc oxide remains. The zinc naphthenate produced is therefore considered to be the neutral form.
- Sample preparation: Sample used for hazard testing was prepared as a neat substance without base oil.

Specific details on test material used for the study:

- Source and lot/batch No.of test material: A036/99
- Expiration date of the lot/batch: 31 August 2018
- Storage condition of test material: Room temperature

In vitro test system

Test system:

human skin model

Source species:

other: Reconstructed human epidermis (RHE)

Cell type:

non-transformed keratinocytes

Cell source:

other: Epiderm Skin Model

Source strain:

other: Epiderm Skin Model

Details on animal used as source of test system:

Not applicable

Justification for test system used:

The test is based on the experience that corrosive chemicals show cytotoxic effects following short term exposure to the stratum corneum of the epidermis. The test is designed to predict and classify the skin corrosion potential of a test item by assessment of its effect on a three-dimensional human epidermis model.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Tissue batch number(s): 27631
- Production date: Not specified
- Shipping date: Not specified
- Delivery date: Not specified.
- Date of initiation of testing: 27/11/17

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ± 1.0°C (actual range 36.2 - 37.3°C).
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C (actual range 36.2 - 37.3°C).

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item.
- Observable damage in the tissue due to washing: None noted
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 5 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570nm
- Filter and bandwidth: Not specified
- Linear OD range of spectrophotometer: Not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA: Not specified

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: Not applicable

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: An excess amount of the solid test item applied directly on top of the skin tissue using a nylon mesh disk. The mesh disk matched the size of the tissue completely, resulting in a fully covered tissue with test item.
- Concentration: Test material was applied neat.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration: Not applicable. Milli-Q water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration: 8N KOH

Duration of treatment / exposure:

3 minutes and 1 hour.

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Run 1 / 3 minute application

Value:

60

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Run 2/ 1 hour application

Value:

83

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test item did not interfere with the MTT endpoint.
Because the mean relative tissue viability for Naphthenic acids, zinc salts was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment Naphthenic acids, zinc salts is considered to be not corrosive.
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit =<2.8) and the laboratory historical control data range. The mean relative tissue viability following the 1-hour exposure to the positive control was 7.6% (acceptance limit =< 15%). In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was =< 28% (acceptance limit =< 30%), indicating that the test system functioned properly.

Any other information on results incl. tables

Table1          
Mean Absorption in the in vitro Skin Corrosion Test with Naphthenic acids, zinc salts

	3-minute application					1-hour application
	A (OD570)	B (OD570)	Mean (OD570)		SD	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative control	1.885	1.816	1.851	±	0.049	2.092	1.724	1.908	±	0.260
Test item	1.184	1.055	1.119	±	0.091	1.540	1.626	1.583	±	0.061
Positive control	0.422	0.304	0.363	±	0.083	0.162	0.128	0.145	±	0.024

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.0414). Isopropanol was used to measure the background absorption.

Table2          
Mean Tissue Viability in the in vitro Skin Corrosion Test with Naphthenic acids, zinc salts

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
Test item	60	83
Positive control	20	7.6

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion the test item is not corrosive in the in vitro skin corrosion test under experimental conditions.

Executive summary:

Eurling (2017) is a GLP-compliant study following the OECD Guideline 431. The study is assigned a Klimisch score of 1 (reliable with restrictions). The mean tissue viability in the in vitro skin corrosion test had a 100% tissue viability for the negative control after a 3 minute and 60-minute application indicated no potential for skin corrosion which was expected. The positive control had a mean tissue viability of 20% for the 3-minute application and 7.6% mean tissue viability after a 1-hour application, which was a positive indication of skin corrosion. The test item had a mean tissue viability of 60% after a 3-minute application and a mean tissue viability of 83% in the 1-hour application. Therefore, the test item to be considered as non-corrosive to the skin.